Mammalian growth factor

ABSTRACT

A homogeneous K-FGF polypeptide having a molecular weight of about 19,000 daltons when analyzed by SDS polyacrylamide gel electrophoresis, wherein the polypeptide is substantially free of oligosaccharide moieties attached to the polypeptide, appears as a single band on SDS-PAGE, exhibits a detectable level of mitogenic activity on growth arrested cells in a  3 H-thymidine uptake assay, and is substantially free from other mammalian proteins, is provided.

This is a continuation, of application Ser. No. 08/056,482, filed May 3,1993, now U.S. Pat. No. 5,750,659 which in turn is a continuation ofSer. No. 07/806,771, filed Dec. 6, 1991, now abandoned, which in turn isa continuation of Ser. No. 07/177,506, filed Apr. 4, 1988, nowabandoned, which in turn is a continuation-in-part of Ser. No.07/062,925, filed Jun. 16, 1987, now abandoned.

The United States Government has rights to this invention by virtue ofgrant No. CA-42568 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

This invention is related to a novel polypeptide having mammalian growthfactor activity and to methods for using it.

A variety of diffusible factors which stimulate the growth of cells in ahormone-like manner are generally called “growth factors”. Growthfactors are present in serum and have also been isolated from a varietyof organs. They are protein molecules (or groups of such molecules) andin all known cases they interact with specific cell surface receptors topromote cellular growth and/or differentiation. Growth factors vary intheir tissue specificity, i.e. some interact only with specific celltypes, while others are active on a wider cell type range.

Among the best known groups of growth factors are: (1) platelet derivedgrowth factor (PDGF), released from platelets; (2) epidermal growthfactor (EGF); (3) hematopoietic growth factors (including interleukins1, 2, and 3), required for growth and differentiation of lymphocytes,and colony stimulating factors (CSF), promoting growth anddifferentiation of hematopoietic stem cells; (4) angiogenic (literally“blood-vessel-forming”) growth factors, such as the fibroblast growthfactors (FGF) believed to promote growth and organization of endothelialcells into new blood vessels; (5) a variety of growth factors releasedby tumor cells and falling into two groups: alpha and beta,corresponding to their chains.

The only well-characterized angiogenic factors are basic and acidicfibroblast growth factors (FGF); believed to be most important in vivofor endothelial cell growth.

It is known that the oncogene that is characteristic of simian sarcomavirus encodes the B chain of PDGF. However, none of the remaining growthfactors mentioned above are produced by oncogenes. Nor do other knownoncogenes produce growth factors.

Growth factors are believed to promote wound healing. For example, EGFpresent in saliva is believed to accelerate wound healing in mice.Schultz G. S et al (Science 232:350-352, 1986) report that transforminggrowth factor (TGF)-alpha and vaccinia virus growth factor (VGF), bothof which are substantially homologous to EGF, accelerated epidermalwound healing in pigs when topically applied to second degree burns andwere significantly more active than EGF.

Of the above-mentioned growth factors, the angiogenic growth factorswould be particularly useful as wound healing agents because of theirability to promote the formation and growth of new blood vessels.Preliminary evidence indicates that the two known (sequenced) angiogenicgrowth factors, basic and acidic FGF (so named due to the total netcharge on the molecules) may be of use as wound healing agents.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a growth factoruseful as a wound healing agent in mammals.

It is another object of the present invention to provide a mammaliangrowth factor with a tissue specificity wider than either acidic orbasic FGF.

Another object is to provide novel pharmaceutical formulations andmethods for promoting would healing.

These and other objects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,accompanying claims and appended drawings.

SUMMARY OF THE INVENTION

The present inventors have unexpectedly discovered a single polypeptidewhich displays substantial homology to each of basic and acidicfibroblast growth factor, said polypeptide having growth factor activityand having an amino acid sequence consisting essentially of the aminoacid sequence of the expression product of a fragment of an oncogeneisolated from Kaposi's sarcoma DNA. In another aspect, the presentinvention is directed to a DNA molecule coding for the abovepolypeptide.

In yet another aspect, the present invention is directed to methods forpromoting the healing of mammalian wounds or burns comprisingadministering to a mammal in need of such treatment a healing-promotingeffective amount of the above polypeptide and to pharmaceuticalformulations comprising said polypeptide and a pharmaceuticallyacceptable carrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the organization of the humanDNA sequences (and probes made thereto) inserted into the mouse genomein the secondary Neo-2 transformant.

FIG. 2 is a schematic representation of the specific region of human DNAsequences shown in FIG. 1 encoding the polypeptide of the presentinvention.

FIGS. 3A and 3B are autoradiographs of Northern blots showing the novelmRNA species encoding the polypeptide of the present invention.

FIGS. 4A-C are schematic representations of the plasmids used in cloningthe genomic DNA fragments and the cDNA encoding the polypeptide of thepresent invention.

FIGS. 5A-D are series of photographs demonstrating the promotion ofgrowth in agar of hamster BHK-21 cells superimposed on a layer of cellstransformed in accordance with present the invention.

FIGS. 6A and 6B are autoradiographs of sodium dodecyl sulfatepolyacrylamide (SDS-PAGE) gels demonstrating the expression of thepolypeptide of the present invention as a fusion protein in bacteria.

FIGS. 7A and 7B are graphs demonstrating the growth-promoting effects ofthe polypeptide of the present invention on the growth of NIH3T3 cells.

FIG. 8 is an autoradiograph of an SDS-PAGE gel demonstrating theproduction of the polypeptide of the present invention by transfectedCOS cells, and its specific immunoprecipitation by rabbit antibodiesdirected against said polypeptide.

FIGS. 9A-C are autoradiographs of SDS-PAGE gels showing the secretion ofthe polypeptide of the present invention in the absence (A) or presence(B) of tunicamycin and of the in vitro translation product (C) which wasimmunoprecipitated by rabbit antibodies directed against the polypeptideof the present invention. Presented in FIG. 9D is the amino acidsequence of the polypeptide showing the sites of glycosylation, cleavageof the signal sequence and potential sites of intramolecular disulfidebonds.

FIGS. 10A-D are autoradiographs an SDS-PAGE gels showing the kinetics ofsecretion of the polypeptide of the present invention, after a 1 hourpulse (A), a 1 hour chase (B), a 7 hour chase (C) or a 19 hour chase (D)and its stabilization by heparin.

FIG. 11 is a graph showing the induction of plasminogen activator bybovine capillary endothelial (BCE) cells treated with the polypeptide ofthe present invention.

FIG. 12 is a bar graph showing the induction of DNA synthesis in BCEcells treated with either basic fibroblast growth factor or thepolypeptide of the present invention.

FIG. 13 is a bar graph showing the effect of the polypeptide of thepresent invention on the proliferation of human umbilical cordendothelial cells (HUVE) in culture and its potentiation by heparin.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have unexpectedly isolated a gene coding for amammalian growth factor. The growth factor gene was produced when theDNA isolated from a Kaposi's Sarcoma (KS) skin lesion, obtained from apatient suffering from AIDS, was transfected into mouse cells. Kaposi'sSarcoma (KS) is a multifocal neoplastic disorder common in patientssuffering from acquired immune deficiency syndrome (AIDS), and alsofound in other immunosuppressed individuals.

The gene of the present invention may be a novel human oncogene and oneof its protein products is significantly homologous to each of the twowell-known angiogenic growth factors, basic and acidic FGF and hasgrowth factor activity. The oncogene which is “activated” in KS cellshas not been heretofore identified.

The gene coding for the growth factor polypeptide of the presentinvention was isolated by transfecting DNA extracted from KS skinlesions of an AIDS patient into mouse NIH3T3 cells (available as ATCCCRL 1658, American Type Culture Collection, Rockville, Md.). Thetransfected cells' ability to produce transformed foci when cultured invitro was then determined.

A transformed focus is a distinct clustering of visually identifiablecells arising from the uncontrolled growth of tumorigenic cells. Anycells capable of taking up and expressing foreign DNA can be employed asthe recipient (the recipient cells are hereinafter referred to asprimary transformants) of the DNA sequences, such as rat F2408, hamsterBHK-21 or preferably mouse NIH3T3 cells.

In a preferred embodiment of the present invention, the DNA sequencesencoding the growth factor can be isolated from the primarytransformants and transferred to normal NIH3T3 cells in a second roundof transfection (hereinafter referred to as secondary transformants)with a selectable genetic marker. The selectable genetic marker can beany gene which confers a selective growth advantage to the recipientcell. Suitable selectable markers include, but are not limited to,resistance to the antibiotic hygromycin, and preferably resistance tothe antibiotic neomycin or G418 (GIBCO, Grand Island, N.Y.). Cells whichare transfected by both the DNA sequence encoding the growth factor ofthe present invention and, for example a gene encoding resistance toG418, can then be selected for their ability to grow in the presence ofconcentrations of about 250 micrograms per ml of G418 present in thegrowth medium.

After the secondary transformants were selected for their ability togrow in the presence of neomycin, or G418 and those cells which had beentransformed to the neoplastic state isolated, the human DNA sequenceswhich had been taken up and integrated into the mouse genome wereidentified, molecularly cloned, using an appropriate vector (EMBL3,described in Frischauf, A. M. et al, J. Mol. Biol. 170: 827-842, 1983)although other vectors could have been used, and mapped by restrictionendonuclease digestion, as detailed in Example 2 below. The sequencesencoded in this DNA region which are transcribed into mRNA (and,presumably into protein) in these cells were identified employing thewell-known Northern hybridization technique with probes obtained byrestriction endonuclease digesting the cloned vector-DNA.

Two unique mRNAs, 1.2 and 3.5 Kb in length were identified, of which the1.2 Kb species encodes the growth factor of the present invention. Thesequence of this 1.2 Kb MRNA is shown in Example 5 below. The 1.2 Kbspecies was found to encode a polypeptide with mammalian growth factoractivity. A comparison of this sequence with the known growth factorsequences revealed that it displayed substantial sequence homology withboth basic and acidic FGF.

In order to determine whether a novel oncogene had been identifiedfollowing the DNA mediated gene transfection, a comparison with otherknown viral and cellular oncogenes was performed.

The transforming DNA sequence m-RNA of the present invention did notdemonstrate any sequence homology to Human Immunodeficiency Virus (thecausative agent of AIDS) or cytomegalovirus DNA as well as herpes virusDNA (viruses which commonly infect AIDS patients cells, the source ofthe DNA sequences of the present invention). In addition, probescorresponding to a number of viral and cellular oncogenes did nothybridize (i.e. no significant sequence homology existed) with thefollowing known oncogenes: three ras oncogenes, myc, sis, erbB, Rel,raf, myb, p53, mos and fos. However, a probe corresponding to theoncogene v-fms (isolated from feline sarcoma virus) revealed a region ofhomology in the cloned (i.e. 32 Kb) genomic DNA sequences. This region(indicated in FIG. 1) was homologous to a portion of the cellular fmsoncogene but it is not transcribed (i.e. these sequences are not presentin the novel mRNA specie described above). Therefore, the fms oncogeneis not responsible for the growth factor activity of the presentinvention. However, the c-fms DNA sequences may contain elements whichactivate the expression of the growth factor sequences in the originalgenomic configuration in the transformants.

Once isolated, the DNA encoding the growth factor of the presentinvention can be cloned and the protein can be expressed in anyeukaryotic or prokaryotic system known in the art. Eukaryotic expressionsystems, such as yeast expression vectors (described by Brake, A. et al,Proc. Nat. Acad. Sci. USA 81: 4642-4646, 1984), Polyoma virus basedexpression vectors (described in Kern, F. G. et al Gene 43: 237-245,1986) or Simian virus 40 (SV40) based expression vectors in COS-1 Simiancells (as described in Gething, M. J. et al Nature 293: 620-625, 1981)are preferred because they are capable of secretion and of performingmodifications (such as glycosylation) necessary for the production ofeukaryotic proteins in their “natural” state, and do so at a highefficiency. Also, the nucleotide sequences of the growth factor of thepresent invention presented in Example 5 below can be used to chemicallysynthesize the gene using techniques known in the art.

The sequence of the expression product of the present invention has beenderived from the DNA sequence. The polypeptide of the present inventioncan be prepared by techniques known in the art. In addition, by routineexperimentation (involving modification of the DNA sequences) theminimum polypeptide sequence having growth factor activity can beidentified. In addition, other modifications to the amino acid sequenceof the present polypeptide may be made provided that they do not affectthe growth factor activity of said polypeptide. The polypeptide of thepresent invention has a sequence that corresponds to the expressionproduct of a fragment of the oncogene from which the present polypeptidewas identified. This is an advantage because the entire expressionproduct of the oncogene need not be produced. Without wishing to bebound by theory, it is believed that the polypeptide of the presentinvention is similar to, if not identical with, its cellularprotooncogene.

The present inventors have also found that the growth factor of thepresent invention stimulated proliferation and plasminogen activatorproduction in endothelial cells in culture. It has also been found thatheparin is required for the above-mentioned effects to be manifested inendothelial cells, but not in cells of fibroblast origin. In human cordvein endothelia, heparin potentiates the effect, but is not essential.It is believed that heparin may protect the mammalian growth factor fromdegradation and/or assist in the formation of a temperature tablecomplex. Therefore, pharmaceutical formulations comprising the mammaliangrowth factor of the present invention may also contain an effectiveamount of heparin or fragments thereof as a stabilizing agent. Theamount of heparin to be added can be obtained by routine experimentationwell known in the art.

Studies described below in Example 9 show that the mammalian growthfactor of the present invention may be provided as a secretedglycoprotein and is processed in mammalian cells so that approximately30 amino acids (representing a signal sequence) are removed in order toform the mature protein. Therefore, the mammalian growth factor can beobtained from the conditioned medium of mammalian cells transfected withthe DNA sequences encoding this glycoprotein, such as COS-1 cellsdescribed below.

The mammalian growth factor of the present invention can be employed asa wound-healing agent for various wounds, such as decubitus ulcers orburns. When employed as a wound or burn healing agent, the growth factorof the present invention may be administered to a mammal in need of suchtreatment orally, parenterally, or preferably, topically, directly tothe affected area in amounts broadly ranging between about 10 nanogramsand about 10 micrograms per dose. The number of treatments and theduration can vary from individual to individual depending upon theseverity of the wound or burn. A typical treatment would comprise 2 or 3applications per day, topically administered directly to the wound orburn.

The growth factor of the present invention can be prepared inpharmaceutical formulations to be used as a wound or burn healing agent.Pharmaceutical formulations comprising the mammalian growth factor ofthe present invention (or physiologically acceptable salts thereof) asat least one of the active ingredients, would in addition containpharmaceutically-acceptable carriers, diluents, fillers, salts and othermaterials well-known in the art depending upon the dosage form utilized.For example, parenteral dosage forms would comprise a physiologic,sterile saline solution. Such formulations may also contain heparin orfragments thereof as stabilizing agents. In a particularly preferredembodiment, the mammalian growth factor of the present invention may bemixed with antibiotic creams (such as Silvadene, Marion Laboratories,Kansas City, Mich., Achromycin, Lederle Laboratories, Pearl River, N.Y.,or Terramycin, Pfipharmecs, New York, N.Y.) well-known in the art.

Although the growth factor of the present invention is particularlyuseful as a wound or burn healing agent, it additionally can be employedas a growth promoting agent for cells in tissue culture and/or as apartial serum substitute. The growth-promoting properties areillustrated in Example-6 below.

The invention is described further below and specific examples which areintended to illustrate the present invention without limiting its scope.

EXAMPLE 1: TRANSFECTION OF NIH3T3 CELLS

High molecular weight DNA was extracted from one KS skin lesion, asdescribed in Delli Bovi, P. et al, Cancer Res. 46: 6333-6338, 1986,(incorporated by reference) and transfected into NIH3T3 cells using thewell-known calcium phosphate precipitation technique (Graham, F. L. etal Virology 52: 456-467, 1973). A distinct focus of highly retractilecells was produced over the background of non-transfected NIH3T3 cellsindicating the presence of transformed cells. To insure the homogeneityof the cell population, cells from the primary focus were recloned inagar suspension medium (Stoker, M. et al Nature 203: 1355-1357, 1964incorporated by reference) since only transformed cells are capable ofsuch growth.

Southern blot hybridization, performed with the Blur-8 plasmid (Jelinek,W. R. et al Proc. Nat. Acad. Sci. USA 77: 1398-1402, 1980 incorporatedby reference), containing DNA sequences representative of the AluIfamily of repetitive DNA (a repetitive DNA sequence present in andindicative of DNA isolated from human cells), revealed that all cellswhich were transformed were capable of growth in agar had acquired humanDNA sequences.

Cells from one agar colony isolated as above, were injected into athymicmice (106 cells per mouse) and two out of three mice developed tumors.DNA from one of the tumors (A15T) was used to transfect NIH3T3 cellstogether with a selectable marker, plasmid pIW3 (Pellegrini, S. et alCell. 36: 943-949, 1984 incorporated by reference) which containssequences conferring resistance to the aminoglycoside antibiotic G418 (aneomycin derivative). Mammalian cells, such as NIH3T3 cells, aresensitive to and are killed by these aminoglycoside antibiotics.However, plasmid pIW3 encodes a gene which allows cells to grow in thepresence of neomycin or G418. Selection for cells resistant to G418revealed the presence of two colonies with transformed morphology, suchas a disorganized piling of cell, and a loss of contact inhibition ofgrowth, while selection for focus formation also resulted in theisolation of two morphologically transformed foci.

DNA from one of the colonies resistant to G418 was used for a thirdcycle of NIH3T3 transfection and again produced a small but significantnumber of AluI positive transformed foci. This demonstrated that thehuman DNA sequences identified and used to transfect NIH3T3 cells werecapable of reproducibly transforming these cells, since the transformedphenotype correlated with the presence of the human AluI repetitive DNAin every stage of the assay.

EXAMPLE 2: MOLECULAR CLONING

A genomic library of DNA extracted from one of the neomycin-resistantsecondary transformants (Neo-2) was constructed after endonuclease MboIpartial digestion and cloned into the EMBL3 lambda phage vector(Frischauf, A. M. et al J. Mol. Biol. 170: 827-842, 1983 incorporated byreference). The library was screened for the presence of recombinantphages containing human AluI repetitive DNA by plating the recombinantphages on a lawn of phage-susceptible bacteria, and allowing them toform plaques of bacterial lysis. Phage DNA was collected from theindividual lysates and transferred to nitrocellulose filters (Schleicherand Schul, Keene, NH) and hybridized with a nick-translated, ³²P-labeledpurified 300 basepair BamHI restriction fragment from plasmid Blur-8 (asdescribed in Maniatis et al, Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Lab, NY, 1982). One recombinant phage (KS-2) wasisolated by this procedure. Hybridization with the Blur-8 AluI plasmidand total mouse DNA revealed that it contained one AluI sequence and twostretches of repetitive mouse DNA sequences and, thus represented one ofthe junctions between mouse and human DNA in the secondary Neo-2transformant.

Several restriction enzyme fragments indicated in FIG. 1 were used toperform two further rounds of screening of the same library byhybridization to the above-mentioned DNA fragments. Several recombinantphages were thus isolated which appeared to span the entire insertion ofhuman DNA into the Neo-2 transformant. The restriction map of the humangenomic DNA sequences present in the transfected cells as reconstructedfrom four overlapping recombinant phages among those isolated is shownin FIG. 1.

In FIG. 1, A, B, C, D, E, F, G, H, and I in the upper part of the figurerepresent DNA fragments derived from the phages shown and used in thecharacterization of these sequences by Southern and Northern blottinganalysis. The interrupted lines indicate mouse DNA. The continuous darklines indicate human DNA. The “V” indicates the regions of joiningbetween mouse and human DNA. Open boxes indicate regions containingmouse repetitive DNA. The hatched boxes indicate the regions containingthe human AluI repetitive DNA sequences. Squiggles indicate theapproximate sites of DNA rearrangements. Restriction sites are indicatedas follows: E, EcoR1; B, BamHI; X, XbaI; S, SailI, Sc, SacI.

The restriction map presented in FIG. 1 encompasses approximately 32 Kb(from the X at approximately 12 Kb to just before the V at the 43 Kbmarker in FIG. 1) of human DNA and contains 3 AluI sequences. SeveralDNA fragments derived from these sequences in FIG. 1 were used todetermine the presence and arrangement of the transfected human DNA inprimary and secondary transformants, as well as in normal human DNA.Southern blot hybridization using these probes revealed that all thesesequences studied were present in the secondary and tertiarytransformants, in the two primary tumors, and also in the DNA isolatedfrom the primary focus (primary transformant). Restriction enzymeanalysis and blot hybridization of the cloned human sequences revealedthat they contained four rearrangements with respect to normal humanDNA, i.e. they were derived from the junction of five DNA fragmentswhich are normally not contiguous in the human genome.

EXAMPLE 3: TRANSCRIPTION OF HUMAN SEQUENCES PRESENT IN NIH3T3TRANSFORMANTS

In order to detect whether the specific human sequences present in thesecondary NIH3T3 transformants were transcribed into mRNAs (andpresumably translated into protein) in the transfected NIH3T3 cells,several of the DNA fragments indicated in FIG. 1 (A through H) were usedas probes in Northern blot hybridization. Total RNA from the secondarytransformants was extracted and purified by the guanidinium-cesiumchloride method as described in Kern, F. G. et al Mol. Cell. Biol. 5:797-807, 1985 incorporated by reference. Poly (A)+RNA was selected(using Hybond m-AP paper, Amersham, Arlington Heights, Ill.), and RNAswere fractionated in the presence of formaldehyde by agarose gelelectrophoresis and transferred to nitrocellulose filters as describedby Maniatis et al. (supra). Nucleic acid hybridization, washing andautoradiography were performed as described in Kern et al (supra). Theresults are shown in FIGS. 3A and 3B.

FIGS. 3A and 3B show the results of the Northern blots using probes Gand H to detect novel mRNA species in transformants by hybridization to1.5 micrograms of poly(A)+RNA prepared from NIH3T3(lane 1); secondarytransformant (designated F1A1) (lane 2); secondary transformant (Neo-2)(lane 3); A15T tumor cells (lane 4); human umbilical vein endothelialcells transformed by SV40 (designated HUVE-SV, lane 5).

Probes A, B, C, D, and E (identified in FIG. 1) did not hybridize withany distinct mRNA species among the poly (A)+RNAs extracted from primaryor secondary transformants. Probes F and G hybridized with two novelmRNA species of about 1.2 and 3.5 Kb (FIG. 3A) in primary and secondarytransformants and also with some larger RNA species of variable lengthin some of the cell lines tested (e.g., lanes 2 and 3). These probes didnot detect any distinct RNA species in normal, non-transfected NIH3T3cells (lane 1) and only a faint band of approximately 4 Kb in length inthe RNA extracted from human endothelial cells (lane 5,). Probe H.recognized only the longer mRNA, but not the 1.2 Kb species (FIG. 3B).Thus, it appeared that the transcribed sequences were restricted toabout 10 Kb of human DNA, and they were expressed in these two novelspecies of mRNA which contain common and unique sequences. Anenlargement of the map of FIG. 1 showing the region encoding thesesequences is shown in FIG. 2 (indicated by the arrow 5′-3′).

EXAMPLE 4: BIOLOGICAL ACTIVITY

To map more precisely the transforming DNA sequences, a 11 Kb fragmentgoing from the left polylinker SalI (at about 28 Kb in FIG. 2) site ofphage WII-1 rightward to the next SalI site (FIG. 2) was subcloned intothe XhoI site of the pCD SV40 expression vector (Okayama, H. et al, Mol.Cell. Biol 3: 280-289, 1983, incorporated by reference), in bothorientations with respect to the SV40 promoter, and the resultingplasmids (pCD6.6A and pCD10A, shown in FIGS. 4A and 4B) were tested fortheir biological activity. Both plasmids produced transformed foci onmouse NIH3T3 cells and rat F2408 cells with an efficiency comparable tothat of a control plasmid (pTB-1) containing an activated ras oncogene(as described in Goldfarb, H. et al, Nature 296: 404-409, 1982incorporated by reference).

Cells (approximately 1×10⁶ per dish) were transfected using the calciumphosphate precipitation technique as described above with the plasmidDNA together with 20 micrograms of mouse carrier DNA. Each culture wasthen subdivided into five plates. Foci were counted at 2-3 weeks aftertransfection. The results are presented below in Table I.

TABLE I Transformation of Mouse and Rat Fibroblasts with RecombinantPlasmid DNAs Foci/microgram DNA Plasmids NIH3T3 Rat F2408 EXPT. I pCD(WII-1) 10 A* 900 192 pCD (WII-1) 10 B* 800 — pGEM (WII-1) 10 120 —pTB-1 (ras) 800 520 EXPT. II pCD (WII-2) 6.6 A* 2500 150 pCD (WII-2) 6.6B* 1400  80 pGEM (WII-2) 6.6 40 — pTB-1 2500 400 EXPT. III pTB-1 500 —p9BKS3A** 2000 — p9BKS3B** <1 — pCD (WII-1) 10 A 1400 — *A and Bindicate the position of the SV40 promoter/enhancer element with respectto the polarity of transcription (going from left to the right inFIG. 1) of the inserted SalI genomic fragments contained in the pCDexpression vector. The “A” constructs have the SV40 promoter/enhancer in5′ position and the “B” constructs in 3′ position. **The p9BKS3 plasmidscontain the cDNA encoding the growth factor of the present invention inthe 5′-3′ polarity (A) or 3′-5′ polarity (B).

As can be seen from Table I above, the same DNA fragment was capable ofproducing transformed foci when inserted into the pCD and pGEM3bacterial vector (the latter available from Promega Biotech, Madison,Wis.), but in the case of the pGEM3 vector, with about 8-fold lowerefficiency.

A 6.6 Kb DNA fragment going from the left SalI site of phage WII-2 tothe same SalI site used for the above-mentioned constructs FIG. 1) wasalso cloned using both pCD and pGEM vectors. The pCD-6.6 constructstransformed both mouse and rat cells with high efficiency similar tothat of pTB-1 and that of the pCD10 plasmids, whereas the pGEMconstructs were transformed with an efficiency about 40 fold lower(Experiment II, Table I). Therefore the 6.6 Kb fragment appeared tocontain all of the sequences encoding a transforming gene and also atranscriptional promoter since it functioned in a plasmid vector devoidof any mammalian transcriptional regulatory elements. The higherefficiency of transformation of the pCD plasmids is probably due to thepresence of the SV40 “enhancer” sequences.

EXAMPLE 5: C-DNA CLONING

To precisely identify the DNA sequences responsible for the growthfactor activity of the cloned DNA products, a complementary DNA (cDNA)library was constructed from the poly(A)+RNA isolated from one of thetransformants (A15T). This library was constructed in a bacteriophagelambda gt10 vector (Huynh, T. V. et al in DNA Cloninq: A PracticalApproach D. Glover, ed. Vol 1: 49-78, Oxford Press, 1985 incorporated byreference), and the recombinant phages plaques (from Example 2) screenedwith the probes G and H (in FIG. 1). The library was constructed using acDNA synthesis system (Amersham Corporation, Arlington Heights, IL) andthe poly(A)+RNA obtained from the AI5T cell line isolated by theguanidiumisothiocyanate procedure as described in Example 3 above.Following methylation with EcoRI methylase and the addition of EcoRIlinkers, the linkers were digested and the cDNA size-fractionated bycolumn chromatography (A50m column, BioRad, Richmond, Calif.). The cDNAwas then ligated to EcoRI digested, dephosphorylated lambda-gt10 arms(Promega Biotech, Madison, Wis.). The ligated cDNA was then packaged(using Gigapack extracts, Stratagene Cloning Systems, San Diego, Calif.)and plated, using C600 Hf1 (E.coli) as a host strain. A cDNAcorresponding to the 1.2 Kb mRNA was isolated by plaque hybridization toprobe G.

Subcloning of the cDNA insert cD3, a clone which contained the cDNAcorresponding to the 1.2 Kb mRNA above, into mammalian expression vector91023B (Kauffman, R. J. Proc. Nat. Acad. Sci. USA 82: 689-693, 1985incorporated by reference) produced plasmid p9BKS3A and its biologicalactivity was confirmed, i.e. it was capable of transforming NIH3T3 cellswith a high efficiency upon transfection (Table I, Expt III). This cDNAwas also subcloned into pGEM-3 sequencing vector (Promega Biotec,Madison, Wis.) and sequenced by the dideoxy method of Sanger, F. (Proc.Nat. Acad. Sci. USA 74: 5463-5467, 1977 incorporated by reference) andin part by the method of Maxam, A. U. and Gilbert, W. (Methods Enzymol65: 499-560, 1980, incorporated by reference). The nucleotide sequenceis presented below.

           10           20            30           40                         *             *            * GG CGC GCA CTG CTCCTC AGA GTC CCA GCT CCA GCC GCG CGC TTT CCG 50            60           70           80            90  *             *            *            *             * CCC GGC TCGCCG CTC CAT GCA GCC GGG GTA GAG CCC GGC GCC CGG GGG    100          110           120          130          140     *            *             *            *            * CCC CGT CGCTTG CCT CCC GCA CCT CCT CGG TTG CGC ACT CCC GCC CGA       150          160          170           180          190        *            *            *             *            * GGT CGGCCG TGC GCT CCC GCG GGA CGC CAC AGG CGC AGC TCT GCC CCC         200           210          220          230          *             *            *            * CAG CTT CCC GGG CGCACT GAC CGC CTG ACC GAC GCA CGC CCT CGG GCC240         250          260           270          280*            *            *             *            * GGG ATG TCG GGGCCC GGG ACG GCC GCG GTA GCG CTG CTC CCG GCG GTC    \Met Ser Gly Pro GlyThr Ala Ala Val Ala Leu Leu Pro Ala Val 290           300          310          320           330  *             *            *            *             * CTG CTG GCCTTG CTG GCG CCC TGG GCG GGC CGA GGG GGC GCC GCC GCA Leu Leu Ala Leu LeuAla Pro Trp Ala Gly Arg Gly Gly Ala Ala Ala    340          350           360          370           380     *            *             *            *            * CCC ACT GCACCC AAC GGC ACG CTG GAG GCC GAG CTG GAG CGC CGC TGG Pro Thr Ala Pro AsnGly Thr Leu Glu Ala Glu Leu Glu Arg Arg Trp       390          400          410           420          430        *            *            *             *            * GAG AGCCTG GTG GCG CTC TCG TTG GCG CGC CTG CCG GTG GCA GCG CAG Glu Ser Leu ValAla Leu Ser Leu Ala Arg Leu Pro Val Ala Ala Gln                       450          460          470          *             *            *            * CCC AAG GAG GCG GCCGTC CAG AGC GGC GCC GGC GAC TAC CTG CTG GGC Pro Lys Glu Ala Ala Val GlnSer Gly Ala Gly Asp Tyr Leu Leu Gly480         490          500           510          520*            *            *             *            * ATC AAG CGG CTGCGG CGG CTC TAC TGC AAC GTG GGC ATC GGC TTC CAC Ile Lys Arg Leu Arg ArgLeu Tyr Cys Asn Val Gly Ile Gly Phe His 530           540          550          560           570  *             *            *            *             * CTC CAG GCGCTC CCC GAC GGC CGC ATC GGC GGC GCG CAC GCG GAC ACC Leu Gln Ala Leu ProAsp Gly Arg Ile Gly Gly Ala His Ala Asp Thr    580          590           600          610          620     *            *             *            *            * CGC GAC AGCCTG CTG GAG CTC TCG CCC GTG GAG CGG GGC GTG GTG AGC Arg Asp Ser Leu LeuGlu Leu Ser Pro Val Glu Arg Gly Val Val Ser       630          640          650           660          670        *            *            *             *            * ATC TTCGGC GTG GCC AGC CGG TTC TTC GTG GCC ATG AGC AGC AAG GGC Ile Phe Gly ValAla Ser Arg Phe Phe Val Ala Met Ser Ser Lys Gly         680           690          700          710          *             *            *            * AAG CTC TAT GGC TCGCCC TTC TTC ACC GAT GAG TGC ACG TTC AAG GAG Lys Leu Tyr Gly Ser Pro PhePhe Thr Asp Glu Cys Thr Phe Lys Glu720         730          740           750          760*            *            *             *            * ATT CTC CTT CCCAAC AAC TAC AAC GCC TAC GAG TCC TAC AAG TAC CCC Ile Leu Leu Pro Asn AsnTyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro 770           780          790          800           810  *             *            *            *             * GGC ATG TTCATC GCC CTG AGC AAG AAT GGG AAG ACC AAG AAG GGG AAC Gly Met Phe Ile AlaLeu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn                 830           840          850          860     *            *             *            *            * CGA GTG TCGCCC ACC ATG AAG GTC ACC CAC TTC CTC CCC AGG CTG TGA Arg Val Ser Pro ThrMet Lys Val Thr His Phe Leu Pro Arg Leu ---       870          880           890          900          910        *            *            *             *            * CCC TCCAGA GGA CCC TTG CCT CAG CCT CGG GAA GCC CCT GGG AGG GCA         920          930          940           950          *             *            *            * GTG CGA GGG TCA CCTTGG TGC ACT TTC TTC GGA TGA AGA GTT TAA TGC960         970          980           990         1000*            *            *             *            * AAG AGT AGG TGTAAG ATA TTT AAA TTA ATT ATT TAA ATG TGT ATA TAT1010          1020         1030          1040         1050  *             *            *            *             * TGC CAC CAAATT ATT TAT AGT TCT GCG GGT GTG TTT TTT AAT TTT CTG   1060          1070         1080         1090          1100     *            *             *            *            * GGG GGA AAAAAA GAC AAA ACA AAA AAC CAA CTC TGA CTT TTC TGG TGC      1110         1120         1130          1140        *            *            *             * AAC AGT GGA GAA TCTTAC CAT TGG ATT TCT TTA ACT TGT

The above nucleotide sequence is unusual in many respects. It isextremely G-C rich (75-85%) in approximately the first 650 nucleotides(5′-3′) while its 3′ part is rich in sequences of the ATTT(A) typecharacteristic of unstable mRNAs. There was only one open reading frame(encoding a protein) with an in-frame ATG (the initiation codon forprotein translation) which would encode a protein comprising 206 aminoacids. Analysis of the predicted protein sequences revealed asignificant (substantial) homology (approximately 45%) to mature bovinebasic as well as human basic FGF as described by Abraham, J. A. et al(Science 233: 545-548, 1986; EMBO J. 5: 2528, 1986). A less stringent(but still substantial) homology (approximately 35%) was noted withrespect to bovine acidic FGF whose sequences was described inGimenez-Gallego, G. et al, Science 230: 1385, 1985. If one takes intoaccount not only amino acid identity, but also conserved substitutions,the deduced homology becomes higher (approximately 65% and 60% for basicand acidic FGF, respectively). The first portion of the growth factor ofthe present invention (approximately 70 amino acids) did not demonstrateany homology to the two FGF primary sequences and strictly speaking maynot be necessary for growth factor activity. This portion contained apossible signal peptide important for secretion of secretory proteins. Acomparison of the protein sequence of the growth factor of the presentinvention and those of bovine basic and acidic FGF is shown below inTable II.

TABLE II BOVINE BASIC FIBROBLAST GROWTH FACTOR   1′MSGPGTAAVALLPAVLLALLAPWAGRGGAAAPTAPNGTLEAELERRWESLVALSLARLPV  61′AAQPKEAAVQSGAGDYLLG-IKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSL-LEL         ..:.:..  : .:  .::::. : :: :.  ::::..:.....   . :.:   1″PALPEDGGSGAFPPGHFKDPKRLYCKNG-GFFLRIHPDGRVDGVREKSDPHIKLQL 119′SPVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGMF  . ::::::::: .. ..::...:.: .:   :::: : : : .::::.: : ::.. .  56″QAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYSSWY 179′IALSKNGKTKKGNRVSPTMKVTHFLPRL .::...:. : : ...:. :.. ::: 116″VALKRTGQYKLGPKTGPGQKAILFLPMSAKS TC,1 BOVINE ACIDIC FIBROBLAST GROWTHFACTOR   1′ MSGPGTAAVALLPAVLLALLAPWAGRGGAAAPTAPNGTLEAELERRWESLVALSLARLPV 61′ AAQPKEAAVQSGAGDYLLGIKRLRRLYCNVGIGFHLQALPDGRIGGAHADTRDSL-LELS                : . :. . :::. : :. :. :::: ..:... . . . :.:.   l″FNLPLGNYKKPKLLYCSNG-GYFLRILPDGTVDGTKDRSDQHIQLQLC 120′PVERGVVSIFGVASRFFVAMSSKGKLYGSPFFTDECTFKEILLPNNYNAYESYKYPGM-- . . : : :....  :.::...: ::::.  ..:: : : :  :.::.: : :....  48″AESIGEVYIKSTETGQFLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAEKHW 178′FIALSKNGKTKKGNRVSPTMKVTHFLPRL :..:.:::..: : :.  . :.. ::: 108″FVGLKKNGRSKLGPRTHFGQKAILFLPLPVSSD A = Ala; R = Arg; N = Asn; D = Asp; C= Cys; Q = Gln; E = Glu G = Gly; H = His; I = Ile; L = Leu; K = Lys; M =Met; F = Phe P = Pro; S = Ser; T = Thr; W = Trp; Y = Tyr; V = Val.

In Table II, two dots between a particular set of amino acid residuesindicate exact identity between the growth factor of the presentinvention and either one of the basic or acidic FGF, and one dotindicates a conservative substitution, e.g. substitution of the sametype of amino acid. In addition, the amino acid sequence of the growthfactor of the present invention is presented as the sequences numbered1′-206′, while the FGF sequences are presented as the sequences numbered1″-146″ and 1″-141″ for basic and acidic FGF, respectively.

EXAMPLE 6: BIOLOGICAL ACTIVITY OF MEDIUM OBTAINED BY THE GROWTH OFTRANSFECTED CELLS

In order to demonstrate that cells transfected with the 1.2 Kb cDNAsequences indeed produce the growth factor of the present invention, themedia obtained from culturing the NIH3T3 transformants were tested forability to stimulate cell proliferation and/or to induce in these cellsproperties typical of transformed cells in vitro (e.g. changes inmorphology, ability to grow in suspension in medium containing 0.34%agar). Medium conditioned by incubating monolayer cultures of twotransformed cell lines (A15T and 91B3-1) for twenty hours in Dulbecco'smodified Eagle's medium (DMEM, GIBCO, Grand Island, N.Y.) plus 0.4%serum was applied to cultures of normal NIH3T3 cells which had beenplated in 0.4% calf serum. The medium caused striking morphologicchanges in the cells, indicative of transformed cells. When the cellnumber was measured after six days in culture, the control cultures(kept in 0.4% serum without any other additions) showed practically nogrowth during this time, while cells incubated in conditioned mediumdoubled in number every 48 hours.

The ability of the cells transfected with the growth factor of thepresent invention to secrete the protein was tested in the followingmanner: NIH3T3 cells transformed with p9BKS3A (shown in FIG. 4C) or theA15T primary transformant cell line were plated on the bottom of a 50 mmpetri dish, allowed to grow until semi-confluent, and then covered with7 mls of agar-containing medium. After this agar layer was hardened, anew thin layer (1.5 ml) of agar medium containing 20,000 normal hamsterBHK-21 cells (available as ATCC CCL 8, American Type Culture Collection,Rockville, MD) was added. In this way the cells attached to the bottomof the dish metabolize and still grow to some extent, but they cannotenter in physical contact with the BHK-21 cells of the upper layer.Controls were BHK-21 cells without a “feeder” layer or NIH3T3 cellstransformed by an “activated” ras oncogene (PTB1), an oncogene known totransform these cells. Plates were incubated at 37° C. for about twelvedays. The results are shown in FIGS. 5A-D.

No agar growing colonies of BHK-21 cells were observed in negativecontrols (FIG. 5A) while the ras-transformed cells induced the growth ofonly about 100 microcolonies (FIG. 5B). However, both the A15T cell lineand cells transformed by p9BKS3A plasmids secreted a factor whichinduced the formation of very large colonies in 50 to 90% of the BHK-21cells present in the agar overlay (FIGS. 5C and 5D).

EXAMPLE 7: EXPRESSION OF THE GROWTH FACTOR OF THE PRESENT INVENTION INBACTERIAL CELLS

To prove conclusively that the growth factor of the present inventionwas encoded in the CDNA sequences (approximately 1.2 Kb in lengthdiscussed above), the sequences were expressed in E. coli under thecontrol of an inducible bacterial expression vector.

The vector used was pEx34C (a derivative of pEx31, described in Strebel,K. et al. J. Virol. 57: 983-991, 1986 incorporated by reference) whichcontains the DNA sequences encoding the N-terminal 99 amino acids of thepolymerase of RNA bacteriophage MS2 under the control of an induciblebacteriophage lambda PL promoter. The vector was cut with restrictionendonuclease BamHI, the ends blunted using the Klenow fragment of DNApolymerase, and the blunt-end was ligated to SmaI-cut cDNA contained inthe pGEM-3 vector. SmaI cuts the cDNA at nucleotide 254 (see sequence)and then downstream in the polylinker region of the pGEM-3 plasmid.Using the pEx34C vector, a fusion gene was constructed which encoded afusion protein of approximately 30,000 daltons, comprising the first 99amino acids of the bacteriophage MS2 polymerase followed by all aminoacids encoded by the cDNA inserted except the first four. Induction ofexpression in the appropriate bacterial host (by raising the temperatureto 42° C., as described in Strebel, et al, supra) resulted in thesynthesis of large amounts of a protein of the expected molecularweight, which was absent under non-induced conditions as evidenced bySDS-PAGE of cell extracts (FIG. 6A, lane 2 at 42° C.).

In FIG. 6A, lanes 1 represent the SDS-PAGE results for bacterialextracts transformed with a vector without the cDNA; lanes 2 representthe results of extracts transformed by a cDNA-bearing vector. The arrownext to the 31 kD markers in FIG. 6A represents the position of thepolypeptide of the present invention.

Since this protein, like many other bacterial fusion proteins wasinsoluble, the insoluble fraction of the protein extract was partiallypurified by extraction with 7M urea. This resulted in a proteinpreparation containing the fusion protein and about seven to eight otherbacterial proteins (see the SDS-PAGE of cell extracts in FIG. 6B, lane2). The fusion protein was estimated to represent about 20% of the totalprotein mass. After dialysis, the entire 7M urea protein extract(containing the growth factor fusion protein) was applied to normalNIH3T3 cells at various concentrations and the cells incubated in DMEMplus 0.5% serum for five days. Two controls were used involving cellswhich either received nothing, (except DMEM +0.5% calf serum) or cellswhich received the insoluble, 7M urea-extracted protein fraction frombacteria expressing only the pEx34C vector (without a cDNA insert). Theresults are shown graphically in FIG. 7A.

In FIG. 7A; the symbol “-” represents no addition of extract, “A”represents addition of cell extracts from cells which received thevector without an insert, “A15” represents addition of conditionedmedium from the A15T-transformed cell line mentioned above; and “C”represents addition of cell extracts from cells transformed with vectorsencoding the fusion protein of the present invention. In FIG. 7A, thestriped bars represent: for A15 a 1 to 2 dilution of the conditionedmedium; for C an amount of bacterial extract estimated to correspond to100 nanograms/ml of the fusion protein, and for A an equivalent amountof bacterial proteins extracted from bacteria expressing the vectoralone. The solid bars in FIG. 7A represent: for A15 a 1 to 4 dilution ofthe conditioned medium, for C an amount of extract corresponding to 40nanograms/ml of the fusion protein, and for A an equivalent amount ofextract from bacteria expressing the vector alone.

While cells in the control cultures did not proliferate appreciably in 5days, cells receiving the fusion protein (containing the growth factor)showed appreciable growth (FIG. 7), in a dose-dependent fashion. Thesedata therefore show that a growth factor gene was isolated and that theprotein it encodes was expressed in bacteria.

EXAMPLE 8: EXPRESSION OF THE MAMMALIAN GROWTH FACTOR IN MAMMALIAN CELLS

The plasmid p9BKS3A, containing the cDNA encoding the mammalian growthfactor of the present invention, was transfected into monkey COS cells.COS-1 cells (Gluzman, Y. Cell. 23: 175, 1981 incorporated by reference)are a line of simian cells which constitutively express the SV40 large Tantigen, and thus, any DNA molecule containing the SV40 replicationorigin (such as plasmid P9BKS3A) introduced into these cells can beamplified and expressed. COS cells are available as ATCC CRL 1650 andATCC CRL 1651 from the American Type culture collection Rockville, Md.).By using such a system, the cDNA encoding the growth factor of thepresent invention was amplified and its gene product was overproduced.

COS cells were plated at 1×10⁶ cells per petri dish and incubatedovernight before transfection. Three micrograms of recombinant plasmidDNA (p9BKS3A, FIG. 4C) were transfected using the DEAE-dextran techniquefollowed by chloroquine treatment (Luthman, H. et al Nuc. Acid Res. 11:1265-1308, 1983) to improve the uptake of the transfected DNA. After 45hours, the transfected cells were labeled with ³⁵S-methionine for twohours (200 microci per ml) and total cell lysates were prepared asfollows. Cells were washed in ice cold STE buffer (15mM NaCl; 10 mM TrispH 7.2; 1 mM EDTA) lysed in RIPA buffer (10 mM Tris pH 7.4; 0.15M NaCl;1% sodium deoxycholate; 1% Nonidet P-40; 1 mM EDTA; 10 mM KCl; 1%APROTININ), the cell lysate was vortexed for 30 seconds and thencentrifuged at 4° C. in a microfuge for 30 seconds. The supernatant wasrecovered and the same number of counts for each sample were analyzed bysodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Theresults are shown in FIG. 8.

In FIG. 8, the first three lanes from the left contain (1) labeled totalcell extract proteins from COS cells not transfected with any DNA; (2)COS cells transfected with the vector without any inserted DNA; (3)contains labeled total proteins from cells transfected with the vectorwith the inserted cDNA encoding the growth factor of the presentinvention. The arrow between molecular weight markers 21 and 31 kD onthe right side indicates the polypeptide of the present invention (veryfaint).

As can be seen from FIG. 8, lane 3 a band of about 24 kD not present inthe control cells, was visualized. The smearing of the band is probablydue to post-translational modifications such as glycosylation. When coscell extracts were reacted with a rabbit antiserum directed against thegrowth factor of the present invention (raised by immunization againstthe bacterial fusion protein), a 24 kD protein was specificallyprecipitated by two different antisera A and B (right hand side of FIG.8, labelled A and B; “N” represents preimmune and “I” represents immuneserum).

Extracts of COS cells (transfected with plasmid p9BKS3A) were alsoapplied to mouse NIH3T3 cells incubated in DMEM plus 0.5% calf serum inorder to probe for growth factor activity. In FIG. 7B the cellsreceived: no addition (−), extracts from non-transfected COS cells(COS), extracts from cells transfected with the vector alone(COS/91023B) or extracts from cells transfected with the vector encodingthe growth factor of the present invention (COS/p9BKS3A). As shown inFIG. 7B, soluble protein extracts of sonically-disrupted, transfectedCOS cells produced a considerable increase in cell number during thefour days of incubation whereas control extracts (from COS cells whichwere not transfected at all) did not.

Example 9: SECRETION OF THE MAMMALIAN GROWTH FACTOR FROM TRANSFECTEDCELLS

To study the processing of the mammalian growth factor of the presentinvention, the COS-1 cells transfected with plasmid p9BKS3A of Example 8were labeled with ³⁵S-methionine (1,200 Ci per mmol, New EnglandNuclear, Boston, Mass.) at 200 microci per ml 48-52 hours aftertransfection in the presence or absence of tunicamycin (Tu, 10micrograms per ml, Calbiochem-Behring, San Diego, Calif.), cell extractsprepared as in Example 8 above), and the cell extracts or the culturemedia were subjected to immunoprecipitation using the rabbit antiserumspecific for the mammalian growth factor of the present inventiondescribed above. The immunoprecipitates were then run on 12.5% SDS-PAGE.The results are shown in FIGS. 9A-C.

In FIGS. 9A-C, N=non-immune (or control) sera; I=immune sera. Present onthe right are molecular weight markers (in kilodaltons, Kd). Present inFIG. 9D is the amino acid sequence of the polypeptide of the presentinvention. Arrows indicate the positions of cleavage of the pre-protein,stars under the sequence indicate potential glycosylation signals anddots indicate potential cysteine residues which could formintramolecular disulfide bonds; the underlined amino acid residues arethe presumptive signal sequence.

As can be seen in FIG. 9A an appreciable proportion of the mammaliangrowth factor of the present invention, migrating with an apparentmolecular weight of 22,000 to 23,000 Daltons, is found in the culturemedium as would be expected for a secreted protein (lane 2) and wasspecifically immunoprecipitated with immune serum.

In addition, the mature growth factor of the present invention asproduced by mammalian cells is a glycoprotein as demonstrated by thefact that incubation of cells with tunicamycin, a specific inhibitor ofN-linked glycosylation, resulted in the production of a protein with areduced apparent molecular weight (approximately 19,000 Daltons) whichwas not efficiently secreted (Lane 2, FIG. 9B). The molecular weight ofthe protein produced in vivo in the presence of tunicamycin was comparedwith that of the protein translated in vitro in order to determinewhether any additional processing of the mammalian growth factor of thepresent invention occurred, such as cleavage of the signal peptide. Themammalian growth factor RNA and an “anti-sense” RNA were transcribed invitro using SP6 polymerase (Promega Biotech, Madison, Wis.), theresulting RNAs translated in vitro using a rabbit reticulocytetranslation system (Promega Biotech), and the product immunoprecipitatedwith rabbit antiserum generated against the mammalian growth factor.

FIG. 9C shows that the primary in vitro translation product of themammalian growth factor obtained from the reticulocyte translationsystem had a molecular weight of approximately 22,000 Daltons, inagreement with that predicted from the amino acid sequence (FIG. 9C,lane 2). This is about 3,000 Daltons higher than the unglycosylatedprotein produced in vivo, indicated that the primary translation productis processed to a mature form lacking approximately 30 amino acidresidues. Neither non-immune serum nor the translation product ofantisense RNA resulted in the immunoprecipitation of any product.

To determine whether the site of cleavage corresponds to the putativesignal peptide, the N-terminus of the secreted mammalian growth factorprotein was determined as described below.

COS-1 cells transfected with plasmid p9BKS3A were grown for 48 h aftertransfection. The cells were washed twice in phosphate-buffered salineand a small volume of media (Earle's MEM, Select Amine Kit, Difco,available from Sigma Chemical Co., St. Louis, Mo.) containing either[³H]leucine or [³H]arginine, replacing the corresponding cold aminoacid, was added and the cells grown for a further 7 h. The media wasremoved, clarified by centrifugation and the mammalian growth factor wasimmunoprecipitated as described in Delli-Bovi et al., Cell 50: 729-737,1987, incorporated by reference. The labeled protein was released fromprotein A-Sepharose 4B beads (Pharmacia Fine Chemicals, Piscataway, NewJersey) by heating at 80° C. for 10 minutes in 10 mM Tris (pH 7.6), 1 mMEDTA, 0.1% SDS. A small aliquot of this material was run on SDS-PAGE andfluorographed to verify its purity. The protein/antibody complex wasprecipitated with trichloroacetic acid to remove SDS, the precipitateresuspended in 50% trifluoroacetic acid, loaded directly onto a proteinsequencer (Applied Biosystems model 470A) and sequenced as described inHewick et al., J. Biol. Chem., 256:7990, 1981, incorporated byreference. Fractions containing labeled residues were aligned with thepredicted amino acid sequence to determine the amino terminal residue.

The results obtained indicated that the mature mammalian growth factorprotein had two possible N terminal amino acids, either Ala31 or Pro32(arrows under sequence in FIG. 9D), and had lost the signal peptide.Therefore, as is the case for normal mammalian secretory proteins, thepre-protein co-translationally entered the endoplasmic reticulum (ER)through the normal secretory pathway where the signal peptide wascleaved at residue 30 or 31, and was glycosylated in the ER and theGolgi apparatus before being finally secreted into the culture medium asa mature protein of either 175 or 176 amino acids. Immunofluorescencestaining of transfected COS-1 cells expressing the mammalian growthfactor of the present invention provided a visual demonstration of thelocalization of this growth factor in the ER and cytoplasm when thecells were made permeable to the antibodies (data not shown). When thecells were fixed with formalin, most of the cross-reacting material wasvisualized on the cell surface (data not shown).

Example 10: SECRETION AND STABILIZATION OF THE MAMMALIAN GROWTH FACTORBY HEPARIN

The time course of the secretion of the mammalian growth factor fromCOS-1 cells transfected with the p9BKS3A expression plasmid wasexamined. Since the biological activity of fibroblast growth factors areknown to be potentiated and/or stabilized by heparin, the effect of thepresence of heparin on the stability of the secreted mammalian growthfactor of the present invention was examined. Cells were pulse-labeledfor twenty minutes with ³⁵S-methionine,washed, and the label chased withan excess of cold methionine in the presence or absence of heparin (45micrograms per ml, Sigma Chemical Co., St. Louis, Mo.). The presence ofthe mammalian growth factor protein contained in the cell extract (C) orthe medium (M) of the transfected cultures was determined by SDS-PAGEafter immunoprecipitation. Presented on the left in FIGS. 10A-D aremolecular weight markers (in kilodaltons, Kd); the large arrow indicatesthe position of migration of the polypeptide of the present invention(approximately 23 Kd). In addition, N=non-immune (control) sera andI=immune sera. It should be noted that in all of the data presented inFIGS. 10A-D, only immune sera was capable of immunoprecipitating anyproduct.

FIG. 10B shows that one hour after the pulse, there was an approximate60:40 partition of the mammalian growth factor between the intracellularand the extracellular fraction, respectively, both in the presence(lanes 4-8) or absence (lanes 1-4) of heparin. After a seven hour chase(FIG. 10C), there was a dramatic difference between the two types ofcultures. In the absence of heparin (lanes 1-4), the mammalian growthfactor of the present invention had practically disappeared from theculture medium (lane 4) and only a small amount of the protein remainedin the cells (lane 2). In the presence of heparin (lanes 5-8), a largequantity of the mammalian growth factor could be detected in the medium.This difference was even more pronounced after a nineteen hour chase(FIG. 10D). No labelled protein was detectable in control cultures(lanes 1-4), while those incubated with heparin (lanes 5-8) stillcontain a significant amount of the factor in the culture medium (lane8). Increasing the concentration of heparin (to 90 micrograms per ml,lanes 9-12) resulted in an even higher stability of the protein (lane12), although this effect was not always reproducible. Thus, thepresence of heparin increased the half-life of the mammalian growthfactor protein after secretion suggesting that it may protect theprotein from protease attack and/or help in the formation of atemperature-stable complex.

The effect of the mammalian growth factor of the present invention onthe growth of bovine capillary endothelial (BCE) cells was examined. Theinduction or plasminogen activator (PA) activity, DNA synthesis and cellproliferation in the presence of the mammalian growth factor of thepresent invention was compared with that obtained with basic fibroblastgrowth (bFGF, Amgen Biologicals, Thousand Oaks, Calif.) as these areactivities known to be affected by bFGF. Plasminogen activator activitywas assayed as described in Gross et al. J. Cell Biol. 95:924-981, 1982(incorporated by reference). Conditioned medium produced by COS-1 cellstransfected with the p9BKS3A plasmid was used as the source of themammalian growth factor of the present invention. This mediumeffectively stimulated PA production in BCE cells if the medium wasassayed in the presence of heparin (FIG. 11). In the absence of heparin,there was practically no stimulatory activity above that obtained bycontrol COS-1 cell condition medium (FIG. 11). Heparin by itself had nostimulatory effect. Neutralizing antibodies to bFGF (as described inPresta, M. et al., Mol. Cell. Biol. 6:4060-4066, 1986) were unable toblock the stimulation of PA production induced by the mammalian growthfactor of the present invention. This result indicated that thestimulatory effect was not due to bFGF which might have been releasedfrom the COS-1 cells.

The culture medium from transfected COS-1 cells was also capable ofstimulating DNA synthesis in growth arrested BCE cells. Confluentmonolayers of BCE cells were maintained for seven days in DMEM plus 5%calf serum. The medium was then replaced with fresh DMEM plus 0.5% calfserum containing various additions detailed below. After 20 hours, thecells were labeled with 1 microCi per ml of methyl-[³H]thymidine (6.7 Ciper mmol; New England Nuclear, Boston, Mass.) for 3 hours. Cells werewashed twice with phosphate buffered saline and the incorporation oflabel into trichloroacetic acid preciptable material was determined.

The results of the above-described experiment are shown in FIG. 12 wherelane 1 is control BCE cells, lane 2, BCE cells plus bFGF (10 ng per ml);lane 3, BCE cells incubated with conditioned medium (CM) from controlCOS-1 cells at a 1:20 dilution; lane 4, CM plus heparan (10 microgramsper ml); lane 5, BCE cells incubated with conditioned medium from COS-1cells expressing the growth factor of the present invention(COS-K-FGF-CM) at a 1:20 dilution; lane 6, BCE cells incubated withCOS-K-FGF-CM plus heparin (10 micrograms per ml).

Conditioned medium, obtained from COS-1 cells expressing the growthfactor of the present invention plus heparin (FIG. 12, lane 5) wasalmost as stimulatory as medium supplemented with bFGF (obtained fromAmgen Biologicals, Thousand Oaks, Calif.), (FIG. 12, lane 2) whilemedium from control cells was non-stimulatory (FIG. 12, lane 3). Theproliferative effect of the mammalian growth factor of the presentinvention was also apparent when the number of cells was determined.

The results presented above show that the mammalian growth factor of thepresent invention acted as a growth factor for capillary endothelialcells in culture.

In addition FIG. 13 shows that the mammalian growth factor could alsopromote growth of the human endothelial cells lining large vessels. Inthis experiment, cultures of human umbilical cord vein endothelial cells(HUVE) were plated on multiwell dishes (each well having a surface ofabout 1.5 cm²) in a culture medium consisting of DMEM/F12 Medium mixed1:1 (GIBCO, Grand Island, N.Y.) supplemented with 20% fetal calf serumand either endothelial cells growth supplement at 120 micrograms/ml(ECGS, Collaborative Research, Bedford, Mass.) or the growth factor ofthe present invention (K-FGF, i.e. conditioned medium from COS celltransfected with the p9BKS3A plasmid diluted to a final concentration of5%) with or without heparin (90 micrograms/ml). The media were changedevery two days and the final cell number determined at day 7.

It can be seen from the data presented in FIG. 13 that the HUVE cellsdid not proliferate in absence of added growth factors (−) or withheparin alone (Hep.). The growth factor of the present invention(K-FGF+HEP) was approximately as effective as ECGS+heparin in promotingproliferation of HUVE cells. Although the growth promoting effect of thepolypeptide of the present invention was decreased in the absence ofheparin, it was still very substantial (K-FGF, FIG. 13).

What is claimed is:
 1. A homogeneous K-FGF polypeptide having amolecular weight of about 19,000 daltons when produced by culturingCOS-1 cells containing the plasmid p9BKS3A under the conditions suitablefor expression of the protein and in the presence of tunicamycin.